|
| Status |
Public on Sep 24, 2023 |
| Title |
hCD4_ACT_rep3 |
| Sample type |
other |
| |
|
| Source name |
Blood-derived CD4+ T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Blood-derived CD4+ T cells
|
| Treatment protocol |
Following CD4+ T cell isolation, cells were seeded at a concentration of 1x106 cells/ml and activated with αCD3/αCD28 activating beads (ThermoFisher Scientific) for 72h at a concentration of 1 bead/cell in the presence of 100 U/ml IL-2 (PeproTech) in RPMI +/+ (except for mock controls). At the day of infection, cells were spinoculated in 96-well V-bottom plates (MilliporeSigma) in 50 μl RPMI+/+ with 100 ng (HIVDFII) of p24 per 1×106 cells with 5×106 cells total per well for 2 h at 2350 rpm (1173 × g) at 37°C. After spinoculation, all cells were returned to culture in the presence of 30 U/ml IL-2. Pre-stimulated CD4+ T cells stayed in αCD3/αCD28 activating beads during spininfection and subsequent cell culture. Samples were subjected to fluorescence-activated cell sorting 4 days post infection.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Corning) at 2000 rpm (~ 850 x g) at RT for 30 min, without brake. PBMCs were immediately processed to isolate CD4+ T cells by negative selection using the EasySep Human CD4+ T Cell Isolation Cocktail (StemCell Technologies) according to manufacturer’s protocol. Purified CD4+ T cells were cultured in RPMI +/+ (supplemented with 10% FBS and 1% Penicillin/Streptomycin) at 37°C, 5% CO2.
|
| Extracted molecule |
other |
| Extraction protocol |
Quantitative RNA and protein expression data were generated using the nCounter Vantage 3D RNA:Protein Immune Cell Profiling Assay (NanoString Technologies) and the nCounter SPRINT profiler (NanoString Technologies), comprising 770 RNA and 30 protein targets as well as positive and negative controls. 100,000 cells were used per sample, which were processed according to the manufacturer’s instructions.
|
| Label |
nanostring reporter probe
|
| Label protocol |
NA
|
| |
|
| Hybridization protocol |
Probe hybridization was performed according to manufacturer's protocol MAN-10066-01_Cell_Surface_Protein_Processing_from_Cell_Suspensions and MAN-10060-03_RNA_Protein_Codeset_Hybridization_Setup
|
| Scan protocol |
Data was acquired on the nCounter SPRINT profiler (NanoString Technologies).
|
| Description |
total RNA and protein
|
| Data processing |
RNA and protein expression values were normalized and analyzed using the nSolver Analysis Software 4.0 and the add-on Advanced Analysis Software 2.0.115 (NanoString Technologies). Samples that did not pass the default control performance and quality parameters were excluded from subsequent analysis.
|
| |
|
| Submission date |
Sep 19, 2023 |
| Last update date |
Sep 24, 2023 |
| Contact name |
Hannah Sabeth Sperber |
| E-mail(s) |
Hannahsperber.7@gmail.com
|
| Organization name |
Essen University Hospital
|
| Department |
Institute for transaltional HIV Research
|
| Lab |
Schwarzer Lab
|
| Street address |
Virchowstr. 171
|
| City |
Essen |
| State/province |
NRW |
| ZIP/Postal code |
45147 |
| Country |
Germany |
| |
|
| Platform ID |
GPL33774 |
| Series (2) |
| GSE243577 |
The Hypoxia-regulated Ectonucleotidase CD73 is a Host Determinant of HIV Latency [Array] |
| GSE244193 |
The Hypoxia-regulated Ectonucleotidase CD73 is a Host Determinant of HIV Latency |
|
